With the recent completion of the human genome sequence, the sequencing of other vertebrate genomes has taken center stage. The sequencing of widely studied model organisms (e.g., mouse, rat, and zebrafish) is providing a valuable resource for future experimentation and important insights into vertebrate biology. Less clear is the relative value of other candidate genomes being considered for systematic sequencing, especially with regard to their potential contribution to the annotation and interpretation of the human sequence. To investigate such issues, we are generating large blocks of orthologous sequence from multiple vertebrates for detailed comparative analyses. Specifically, the same targeted genomic regions from multiple vertebrate specie are being isolated in large-insert clones and then sequenced. Efficient methods for designing orthologous hybridization probes and isolating bacterial artificial chromosome (BAC) clones from the different species have been developed and implemented. Following characterization by several mapping methods, tiling paths of BACs are then selected and systematically sequenced. In total, >200 Mb of comparative sequencing data is being generated each year. The establishment of this comparative sequence resource is facilitating the development of new computational tools for multi-species sequence comparisons, providing insight about the appropriate degrees of sequencing finishing that should be pursued in the sequencing of other vertebrate species, and revealing the benefits of sequencing species from a range of evolutionary distances. These efforts are being extensively focused on the recently launched ENCODE project, which aims to identify all functional in a targeted 1% of the human genome.